In biological phenomenon analysis at a cellular level and diagnosis of a disease at a molecular level, it is necessary to detect a specific protein or a specific nucleic acid sequence, and fluorescence is used widely for the detection. Specifically, a method is known that uses a fluorescent substance whose fluorescence intensity increases in response to binding to a target protein and an increase of a target nucleic acid sequence. Representative examples of the fluorescent substance include a method utilizing Foerster resonance energy transfer (FRET) and a substance that intercalates into a double helix structure and emits fluorescence by irradiation with excitation light.
However, there is a possibility that a conventional fluorescent substance emits fluorescence even when it is not bound to a target substance, for example. For the purpose of quenching fluorescence of only an antibody or a nucleic acid sequence labeled with a fluorescent substance, the method utilizing FRET is effective (e.g., Non-Patent Documents 1 to 4). However, making use of FRET requires, for example, the introduction of two types of fluorescent dyes and a unique sequence and the precise design of the position to which each fluorescent dye is bound, which poses the problems of, for example, sequence restriction and manufacturing cost.
Hence, for solving the aforementioned problems, fluorescence detection systems using only one type of dye have been proposed, and the one of them is a complex labeling substance having, as a characteristic chemical structure, a chemical structure in which at least two dye molecules are contained in one molecule, with the at least two dye molecules not exhibiting fluorescence emission due to the excitonic effect obtained when they aggregate in parallel to each other, but exhibiting fluorescence emission with the aggregation state being resolved when the molecules undergo intercalation into or groove binding to nucleic acid (Patent Document 1). Use of the labeling substance of this type as a primer or a probe (e.g. exciton oligomer) obtained by introducing the labeling substance into oligonucleotide for, for example, the amplification and detection of a target nucleic acid is disclosed (Patent Document 2). Note here that, hereinafter, the probe may be referred to as the “exciton probe” or the Eprobe”. This exciton oligomer or the like allows fluorescent switching before and after hybridization with one type of dye; and in the case where the excitonic oligomer or the like is used for real-time monitoring of an amplification reaction, since it gives a sequence specific fluorescent signal, the conventional problem that non-specific amplification is also detected when SYBR green I is used can be overcome. Furthermore, since a fluorophore can be introduced into dT or dC, the restriction of sequence almost can be avoided.